The long-term goal of this work is to change the phenotype of a patient with sickle cell disease (SCO) via genetic modification to an asymptomatic state, such as those associated with the persistence of fetal hemoglobin or with sickle cell trait. Studies during the previous project period have developed and in vitro culture system to test lentiviral (LV) transfer of globin genes to human red cells, and demonstrated the efficient expression of p globin in thalassemia RBC after LV vector transduction of stem cells. This project will use RNA interference and a lentiviral (LV) vector containing novel y-globin or modified p-globin constructs for correction of globin gene production in SCD. The central hypothesis is that LV vectors carrying shRNA against (:!s-globin, along with y or (i globin will correct sickling at low vector copies. In aim 1, antisickling globin LV vectors will be optimized for efficacy. In aim 2, the safety of the globin LV vector and RNAi delivery will be defined. Self-inactivating (SIN) LV globin vectors are among the safest designs of integrating vectors, since they lack of viral enhancers and express globin genes upon erythroid differentiation. Preclinical studies of the safety of SIN LV globin vectors are essential. Integration site analysis and preclinical murine gene transfer studies will be performed to ensure the long-term safety of gene transfer. Safety of delivery of shRNA will be analyzed in primary human cells in vitro, and in sickle mice in vivo. In aim 3, critical clinical and preclinical studies necessary for developing a phase l/ll gene therapy trial will be performed, and the feasibility of apheresis of peripheral blood stem cell collection from non mobilized peripheral blood of SCD patients compared to a bone marrow harvest to collect sufficient HSC for a clinical grade gene transfer. In parallel, we will develop a phase l/ll clinical trial. These studies will build on outstanding infrastructure for gene therapy clinical trials developed at Cincinnati Children's Hospital Medical Center to translate a successful genetic correction of sickle cell disease. Lay summary: A permanent genetic correction of sickle blood stem cells using lentiviral vectors can raise the fetal or normal hemoglobin, and lower sickle hemoglobin to reverse sickling. To develop a clinical trial, this project proposes critical studies to optimize a viral delivery system, to ensure its safety, to collect and isolate sufficient sickle blood stem cells for gene transfer, and to affect their genetic correction.